High density lipoproteins (HDL) and their major protein apolipoprotein A-l (apoA-l) are thought to protect against atherosclerotic cardiovascular disease at least in part by promoting reverse cholesterol transport (RCT), whereby excess cholesterol is removed from the periphery (such as the vascular wall) and returned to the liver for excretion. The broad goal of this project is to generate greater understanding of the molecular physiology of HDL metabolism as it relates to reverse cholesterol transport (RCT) by using integrated in vivo models in mice and extending experiments where possible into humans. It has become clear that the plasma concentration of HDL cholesterol (HDL-C) is not an adequate surrogate for anti-atherogenic effects, and that measures of cholesterol flux through the RCT pathway and of HDL function are more critical with regard to effects on cardiovascular disease. Specific Aim 1 will involve the use of mouse in vivo experiments to study the structure-function properties of apoA-l, in studies that complement the physicochemical studies in Project 2 and the cell-based studies in Project 1. ApoA-l variants will be inserted into AAV8-based gene expression vectors, which will be used to express these variant apoA-l molecules in apoA-l knockout mice and effects on HDL metabolism, RCT, and in selected cases, atherosclerosis will be determined. Specific Aim 2 will address, using mouse models, the roles of lecithin:cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), and phospholipid transfer protein (PLTP) in modulating the rate of macrophage RCT and the relationship to atherogenesis. Specific Aim 3 will extend concepts and approaches developed in mice into the human setting, with the specific goal of measuring RCT using new methods in humans with Tangier disease (ABCA1 deficiency) and with LCAT deficiency. While high levels of HDL cholesterol are associated with reduced risk of coronary artery disease, it is not clear that simply raising levels of HDL cholesterol are sufficient to reduce cardiovascular risk. The mechanisms by which HDL is altered and the impact on the process of reverse cholesterol transport is likely to be a more important determinant of cardiovascular risk. This project will seek to understand the regulation of reverse cholesterol transport in living mice and humans.